The biosynthesis of proteins in genetically altered procaryotic cells generally results in expression of a protein having a methionine attached at the amino terminus. Since the addition of methionine to the native protein may alter its biological activity, conformational stability, antigenicity, etc., it is most desirable, if possible, to remove it.
By insertion of a cleavage site between the amino-terminal methionine and the desired native peptide, one in theory has a greater degree of flexibility in the methodology selected for achieving production of the desired native peptide. In fact, however, there have been only a very limited number of practical methods for achieving selective cleavage.
For example, in those native proteins in which methionine is not present, cyanogen bromide mediated cleavage (methionine being the selective cleavage site) has proven to be a very effective method for generating the native protein. In fact, however, the absence of methionine is a rare occurrence in moderately-sized peptides and proteins. Furthermore, the serious toxicity of cyanogen bromide makes its use less than desirable and necessitates extreme caution in its handling. A most important objective thus is to discover alternate methods by which the amino-terminal methionine can be eliminated with generation of the native biosynthetically-produced protein.
One method recognized to be available for N-terminal methionyl removal involves the use of cathepsin C, also referred to as dipeptidyl-aminopeptidase 1 (DAP-1). Cathepsin C is an enzyme which removes two amino acids, as a unit, from the amino terminus of a protein or polypeptide. Under appropriate conditions, dipeptide removal will commence and continue unless and until (1) the amino group of the N-terminus amino acid is blocked, (2) the site of removal is on either side of a proline, or (3) the N-terminus amino acid is lysine or arginine.
Cathepsin C thus may have some usefulness in producing a desired biosynthetically-produced protein from a precursor molecule by designing such precursor molecule to contain an amino acid residue between the initiating methionine and the desired product. Treatment of the precursor molecule with cathepsin C can result in removal of the initiating dipeptide with production of the desired product. This process, however, may be severely limited in its application since dipeptide removal will be expected to continue sequentially until one of the aforedescribed termination sequences is reached. Thus, the cathepsin C approach should find limited usefulness, being applicable generally only in those instances in which the N-terminal portion of the desired product is itself a cathepsin C stop point.
It has now been discovered, however, that the identity of the N-terminal dipeptide of the precursor molecule plays a role in the degree to which dipeptide removal using cathepsin C can be controlled. It is this finding which forms the basis of this invention. Thus, it has been discovered that, when the initiating dipeptide is Met-Tyr or Met-Arg, dipeptide removal can be carefully controlled to obtain the desired product without further degradation occurring irrespective of whether the dipeptide next in the sequence is a cathepsin C stop point. It is to this discovery that the present invention is directed.